A neurosphere is a culture system composed of free-floating clusters of neural stem cells. Since neural stem cells are not yet studied in vivo, neurospheres provide a method to investigate neural precursor cells in vitro. Putative neural stem cells are suspended in a medium lacking adherent substrates but containing necessary growth factors, such as epidermal growth factor and fibroblast growth factor. This allows the neural stem cells to form into the characteristic three-dimensional (3D) clusters.
When neurospheres are placed in a co-culture alongside muscle cells and under appropriate conditions, an axon may form between the neurospheres and the muscle cells. However, existing three-dimensional assay systems lack means for (i) precise positioning of the motor neuron containing neurospheres with respect to the muscle bundle, which results in significant sample-to-sample variation; and for (ii) compartmentalization, which limits the ability to visualize axon outgrowth or supply or stimulate each tissue type selectively.